J Histochem Cytochem. ciliogenesis in cancer cells, and warrant further investigation of their antineoplastic properties. 0.05, ** 0.005, *** 0.0005 as compared to control. Open in a separate window Figure 5 Confocal fluorescence microscopy images of primary cilia in CFPAC-1 cells treated with selected compoundsCilia were stained with an antibody against acetylated tubulin (green) (A) or with an antibody against IFT88 (red) (B). Nuclei were visualized by staining with DAPI (Blue). Images were captured using Bio-Rad Radiance confocal microscope through a 40X objective lens at 2.3X zoom. The scale bar represents 20 m. Images were processed manually to optimally visualize cilia. Identified ciliogenic drugs induce cilia in multiple cancer cell models To corroborate the ability of these compounds to induce cilia in cancer cells, we tested a selection of the most potent compounds (Clofibrate, Gefitinib, Sirolimus, Imexon and Dexamethasone) in a panel of human cell lines representing different cancer types: A549 (lung cancer), UMRC2 (kidney cancer), SUM159 (breast cancer) and L3.6 (pancreatic cancer) cell lines. As shown in Figure ?Figure6,6, all compounds significantly increased the percentage p-Coumaric acid of ciliated cells in all the four cell lines. These results confirm the potential of the identified compounds as cilium inducers in cancer cells. Open in a separate window Figure 6 Effect of a selection of compounds on ciliogenesis in different LATS1 cancer cell line models as assessed by confocal fluorescence microscopy analysis(A) Quantification of the percentage of ciliated cells. Data are presented as mean (100C300) SEM, * 0.05, ** 0.005, *** 0.0005 as compared to control. (B) Representative images showing the effect of selected compounds on ciliation in different cancer cell line models. All Images were captured using p-Coumaric acid Nikon C2 Eclipse Ti-E confocal microscope at 1.0X zoom using a 60x objective p-Coumaric acid lens. Ciliogenic drugs attenuate cell proliferation at least in part through induction of the primary cilium As the presence of the primary cilium is dependent on the cell cycle and is most prominent in the G0/G1 phase, we examined the effect of the selected drugs on the cell cycle using FACS analysis. Although under the culture condition that we used, cultures were not highly proliferative even in control conditions, most compounds resulted in a further increase in the percentage of cells in the G0/G1 phase, indicative of a further induction of growth arrest (Figure ?(Figure7A).7A). In line with these findings, most compounds attenuated cell proliferation as assessed by a spheroid formation assay of L3.6 cells (Figure ?(Figure7B)7B) and BrdU incorporation in CFPAC-1 cells (Figure ?(Figure7C).7C). To explore to what extent primary cilium occurs secondarily to the growth arrest or in fact actively contributes to the observed attenuation of cell proliferation, we assessed the effect of these compounds on cell proliferation in the presence of the deciliation agent chloral hydrate, which p-Coumaric acid completely removed the cilium (Figure ?(Figure7C).7C). Interestingly, in most cases deciliation largely restored cell proliferation of compound-treated cells (Figure ?(Figure7C),7C), indicating that the antiproliferative effect of these compounds is at least in part caused by their ability to induce the primary cilium. Open in a separate window Figure 7 Anti-proliferative effect of ciliogenic compounds and involvement of the p-Coumaric acid primary cilium(A) Changes in cell cycle profile as determined by FACS analysis of CFPAC-1 cells upon treatment with a selection of ciliogenic compounds. Data are presented as mean SEM, * 0.05, ** 0.005, *** 0.0005 as compared to control. (B) Effect of selected compounds on spheroid formation of L3.6 pancreatic cancer cells. Data are.
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